Fig 3.

Mapping of the ZAP70 promoter: (A) 11 promoter construct was generated and cloned into pGL4.17 luciferase vector; schematic representation shows the position and numbering of the sequence for particular constructs. (B) Expression of ZAP70 protein was determined in Jurkat T cells (J) and Nalm6 (N6) cells using a western blot. β-Actin was a loading control. Gels shown are representative of tri-replicate experiments. (C) 1 μg of each of the 11 ZAP70-promoter-luciferase reporter constructs, or empty vector, was transfected into Jurkat cells along with 100 ng of pRLTK using electroporation. The cultures were harvested 48 hrs after the transfection and luciferase assays were performed. Luciferase activities were normalized to pRLTK activity and expressed as the mean ± S.E. of four independent transfection experiments. (D) ZAP70-promoter-luciferase reporter constructs were transfected into Nalm6 cells and harvested as described in (C). (E) Schematic presentation of four primer pairs (R1–R4) designed to span ZAP70 regulatory region as determined in (C) and (D).